How to synthesize a sequence-specific sgRNA for experiments using EnGen® Sau Cas9
Single guide RNAs (sgRNAs) can be synthesized from a variety of double stranded DNA templates. This protocol contains guidelines for the design of sgRNA from a DNA template (annealed oligos, PCR products, synthetic double-stranded DNA templates).
sgRNAs can be synthesized in vitro for use with EnGen® Sau Cas9 using:
- HiScribe™ T7 High Yield RNA Synthesis Kit (NEB #E2040) or
- HiScribe™ T7 Quick High Yield RNA Synthesis Kit (NEB #E2050)
- Select a 20-22 nucleotide target sequence.
The target sequence is based on a specific region that you are trying to target. An online selection tool such as ChopChop, Benchling or Desktop Genetics can also be used to determine a target sequence.
The 20-22 nucleotide target sequence should be directly upstream of the PAM sequence.For Sau, the PAM sequence is 5’ NNGRRT 3’, where N = any nucleotide and R = A or G.The PAM sequence is required for Cas9 recognition but is NOT part of the sgRNA.
Here is an example:
A potential target sequence (with PAM underlined):
5’ ATCGGAACATGGACTGGACTTCGGAT 3’
- Remove the PAM sequence as it will not be included in the sgRNA sequence:
5’ ATCGGAACATGGACTGGACT 3’
- For transcription using T7 RNA Polymerase, it is important to have a G in the first position of the target sequence. If there is not a G at this position, add a G to the 5’ end of your target sequence.This sequence will now be 21 nucleotides.
5’ GATCGGAACATGGACTGGACT 3’
- Add the T7 promoter sequence (5’ TTCTAATACGACTCACTATA 3’) upstream of the 20 (or 20 + G) nucleotide target-specific sequence.
5’ TTCTAATACGACTCACTATAGATCGGAACATGGACTGGACT 3’
- Add the following DNA sequence to the 3’ end of the T7 promoter/target-specific sequence. This is the sequence that will be recognized by the Sau Cas9 protein:
The underlined G indicates the start of transcription.
- A complementary DNA oligo of the same length can be ordered and annealed to the oligo described above.The resulting double-stranded DNA would serve as the template for T7 in vitro transcription.
- Once the dsDNA template is ready, in vitro transcription can be performed using either the HiScribe™ T7 High Yield RNA Synthesis Kit (NEB #E2040) or the HiScribe™ T7 Quick High Yield RNA Synthesis Kit (NEB #E2050), according to the manual.
The final sgRNA sequence will be:
Alternatively, a synthetic sgRNA could be ordered from an RNA synthesis company.
Please note: These oligos are longer than standard synthesis and may only be offered by certain companies.Alternatively, some companies offer double-strand DNA fragment synthesis service, though the amounts delivered may be low and only enough for a few rounds of in vitro transcription.A PCR step may be included to amplify more dsDNA template for in vitro transcription.